Salmonella Group O antibody (ab78863)

Salmonella Antisera Agglutination Protocol

PRINCIPLE OF MEASUREMENT
When this reagent is mixed with a Salmonella strain which has antigens corresponding to the reagent, an antigen antibody reaction occurs to produce agglutination. This reaction is macroscopically observed to determine each serotype

PROCEDURES

1. Material required but not provided
   Glass slide, Glass pencil, small test tubes, pipette and micropipette, Microbiological loop, physiological saline, physiological saline containing 1vol% formalin, Water bath (50^oC), Autoclave (121^oC) or water bath (100^oC), Centrifuge.
2. Preparation of reagents
   The antisera are ready for use.
3. Specimen
   Cultures of organisms which are derived from a pure culture and identified as Salmonella by biochemical tests should be serotyped. If the specimen consists of multiple strains, the serotype may not be correctly identified. For determination test of the H type, motile strains should be used.
4. Procedures
   A) Slide agglutination for O antigen and Vi grouping
   1. Suspend a certain amount of bacterial growth (3-5 times the amount of a match head) in 0.5ml physiological saline and use as antigenic suspension for group O.
   2. Place a drop of polyvalent antiserum and physiological saline (30 µl) as a control onto a clean glass slide partitioned into several parts with a glass pencil.
   3. Place an antigenic suspension for group O (5 - 10 µl) onto the serum and physiological saline on the glass slide.
   4. Mix the reagents by tilting the glass slide back and forth for 1 minute and the agglutination pattern is observed. Agglutination is grossly observed with light through the slide using fluorescent light. It should be first confirmed that no agglutination is found on the reaction with antigenic suspension and physiological saline. Only strong agglutination observed within 1 minute in the reaction with each serum should be regarded as positive. Delayed or weak agglutination is regarded as negative.
   5. If a specimen tests positive with a polyvalent serum, perform steps 2-4 above using each monovalent serum in the polyvalent serum showing positive.
      
      When no agglutination is found with both O and O1 polyvalent sera, repeat steps 2-4 above using Vi serum. If a positive reaction is found with the Vi serum, perform steps 6-8 below.
      
      
   6. Add 0.2ml antigenic suspension for O group into 2ml physiological saline and heat to 121^oC for 15 minutes or 100^oC for 1 hour. Centrifuge the heated solution at 900 g for 20 minutes, discard the supernatant, resuspend the precipitate with 0.2ml physiological saline and use as a heated cell suspension.
   7. Perform steps 2-4 using polyvalent sera and Vi sera with heated cell suspension.
   8. If the live cell shows a negative result with the polyvalent sera and a positive result with the Vi sera, while the heated cell shows a positive result with polyvalent sera and negative result with Vi serum, the specimen is probably regarded as S.Typhi or S.Paratyphi C. Repeat the agglutination test using group O7 and group O9 sera with heated cell suspension.
   
   B) H antigen serotyping
   a) Tube agglutination test for H antigen serotyping
   1. Using a liquid culture of organisms grown at 37^oC for 6-8 hours, make a 1/2 dilution by adding an equal volume of physiological saline containing 1vol% formalin and use as antigenic suspension for H types.
   2. Add three drops of required type of H serum and 100 µl physiological saline into small test tubes respectively, and add 0.5ml antigenic suspension for H type to it.
   3. Mix the contents of the test tube by shaking thoroughly and allow to stand in a water bath at 50^oC for 1 hour, and observe agglutination. Agglutination is grossly observed under sufficient halation of a fluorescent light. It should be first confirmed that no agglutination is found in the reaction with antigenic suspension and physiological saline. Cotton-wool-like agglutination observed after the reaction with each serum should be regarded as positive. If equal suspension is still observed it should be regarded as negative.
   4. If a specimen tests positive with H-L, H-1, H-G, H-z4, H-e, n perform steps 2-3 above using each H factor serum.
   
   b) Slide agglutination test for H antigen serotyping
   1. Suspend a certain amount of bacterial growth (3-5 times the amount of a match head) in 0.5 ml physiological saline and use as antigenic suspension for H serotyping.
   2. Place a drop of H-serum and physiological saline (30 µl) as a control onto a cleaned glass slide partitioned into several parts with a glass pencil.
   3. Place an antigenic suspension for H serotyping (5-10 µl) onto the serum and physiological saline on the glass slide.
   4. Mix the reagents by tilting the glass slide back and forth for 1 minute and the agglutination pattern is observed. Agglutination is grossly observed under transmitted light including fluorescent light. It should be first confirmed that no agglutination is found in the reaction with antigenic suspension and physiological saline. Only strong agglutination observed within 1 minute in the reaction with each serum should be regarded as positive. Delayed or weak agglutination is regarded as negative.
   5. If a specimen tests positive with H-L, H-1 , H-G, H-z4, H-e, n serum, perform steps 2-4 above using each H factor serum. If specimens tests positive with the polyvalent serum perform steps 2-4 above using each H serum in the polyvalent serum showing positive.

1. Bacterial culture should be performed using non-selective media e.g. nutrient agar. If selective media are used, antigen production may be insufficient or autoagglutination may occur.
2. When antigenic suspension and serum are mixed as a procedure of slide agglutination, the microbiological loop should be sterilized with a flame for each serum to avoid cross-contamination among sera.
3. When the H type of a strain with weak motility is determined, the strain should be passed through a semi-liquid medium inserted into a Craigie's tube before determination to enhance the motility.
4. When agglutination is found in the reaction of antigenic suspension and physiological saline, the determination test is repeated after a colony is reselected.
5. When both O and O1 polyvalent sera give positive results, reconfirm the biochemical properties. If the strain is identified as Salmonella, it may possess other O antigens that are not included in Salmonella antisera.
6. If the strain is identified as Salmonella and tested negative for polyvalent sera and Vi serum, it may possess O antigen that is not included in Salmonella antisera .
7. If heated antigenic suspension after heating and centrifugation twice tests positive for Vi serum and negative for O polyvalent sera, its biochemical properties should be reconfirmed.
8. Some strains of Citrobacter and Escherichia are known to possess Vi antigen. If a viable organism suspension tests positive for Vi serum and heated antigenic suspension tests negative for Vi sera and O polyvalent serum, then the biochemical properties should be reconfirmed.
9. As aggregate by the reaction of flagella is very fragile, the test tube should not be shaken during the observation. If agglutination is indistinct after an hour of reaction it should be determined after 1 more hour.
10. If the specimen tests negative with all of the H sera, the strain of specimen may have a serum type other than the tested types or the flagella of the strain may not have grown sufficiently, The determination test of the H type should be repeated for confirmation after mobility enhancement procedures.

1. Sensitivity test
   a) O sera: When one drop of the product reacted on a glass slide with a reference strain of a known serotype granular agglutination was grossly observed.
   b) H sera: When 3 drops of the product reacted in a small test tube with a reference strain of a known serotype, cotton-wool like agglutination was grossly observed.
   c) Vi serum : When one drop of the product reacted on a glass slide with a reference strain of a known serotype, granular agglutination was grossly observed.
2. Specificity test
   In test performed in a similar manner to the sensitivity test the antiserum agglutinates only with the reference strain corresponding to the serotype, while in reactions with non-corresponding reference strains, macroscopic agglutination is not observed.